Drugs in the Pipeline for HBV

Open AccessPublished:May 29, 2019DOI:https://doi.org/10.1016/j.cld.2019.04.006

      Keywords

      Key points

      • The primary goal of current therapeutic research for chronic hepatitis B is to achieve a functional cure after a finite course of therapy.
      • Both direct-acting antivirals, targeting different aspects of the hepatitis B virus (HBV) replication cycle, and immunotherapeutic approaches are being explored as monotherapies and/or in combination with other agents. The key molecular objective is elimination of HBV covalently closed circular DNA, which is the source of viral transcripts associated with chronic infection, but minimally affected by current therapies.
      • Direct-acting antivirals under clinical investigation include entry inhibitors, core protein inhibitors, and RNA silencers.
      • Immunotherapeutic approaches include TLR-7 and TLR-8 agonists, therapeutic vaccines, checkpoint inhibitors, RIG-I agonist, and anti-HBV antibodies.
      • Results of preclinical and early clinical studies are promising; in the next few years, anticipated phase 2 and phase 3 data will establish which drugs or drug combinations may contribute to a functional cure.

      Introduction

       Global Burden

      Despite the introduction of an effective vaccine almost 40 years ago, hepatitis B virus (HBV) remains a major cause of chronic liver disease. Globally, an estimated 257 million people have chronic hepatitis B (CHB), and 600,000 to 1 million deaths occur annually because of the end-stage complications of cirrhosis and hepatocellular carcinoma.
      • World Health Organization
      The global burden of disease attributable to chronic viral hepatitis (B and C combined) has increased over the last 2 decades. Viral hepatitis was the seventh leading cause of death worldwide in 2013, compared with tenth in 1990.
      • Stanaway J.D.
      • Flaxman A.D.
      • Naghavi M.
      • et al.
      The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013.

       Hepatitis B Virus Life Cycle

      The complex HBV life cycle has recently been extensively reviewed and presents multiple potential antiviral targets.
      • Seeger C.
      • Mason W.S.
      Molecular biology of hepatitis B virus infection.
      • Ko C.
      • Michler T.
      • Protzer U.
      Novel viral and host targets to cure hepatitis B.
      Replication begins when circulating HBV binds to a hepatocyte through interactions between hepatitis B surface antigen (HBsAg) and sodium taurocholate cotransporting polypeptide (NTCP) as a receptor.
      • Seeger C.
      • Mason W.S.
      Sodium-dependent taurocholic cotransporting polypeptide: a candidate receptor for human hepatitis B virus.
      • Yan H.
      • Zhong G.
      • Xu G.
      • et al.
      Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
      Subsequently, the viral capsid is released into the cell and traffics to the nuclear pore, where the HBV relaxed circular DNA (rcDNA) can be delivered to the nucleus. In the nucleus, the rcDNA is converted by host polymerases to covalently closed circular DNA (cccDNA), which is a nuclear mini-chromosome-like moiety that exists in low copy numbers in infected hepatocyte nuclei.
      • Qi Y.
      • Gao Z.
      • Xu G.
      • et al.
      DNA polymerase κ is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B virus.
      cccDNA is thought to be relatively stable in quiescent hepatocytes but may be lost during cell division.
      • Dandri M.
      • Petersen J.
      Mechanism of hepatitis B virus persistence in hepatocytes and its carcinogenic potential.
      • Yang H.C.
      • Kao J.H.
      Persistence of hepatitis B virus covalently closed circular DNA in hepatocytes: molecular mechanisms and clinical significance.
      cccDNA molecules function as templates for transcription of full-length pregenomic RNA (pgRNA) and subgenomic messenger RNAs (mRNAs) that encode viral proteins. These proteins include HBV polymerase/reverse transcriptase (pol/RT), hepatitis B x-protein, core protein, hepatitis B e-antigen (HBeAg), and HBsAg. HBV pol/RT binds to pgRNA and is encapsidated within 120 core protein dimers to form a nascent viral capsid, where the pgRNA is reverse transcribed to produce viral rcDNA. The capsid can then either acquire an HBsAg-containing envelope and be secreted as infectious virus or traffic back to the nucleus and replenish the cccDNA pool.
      • Ko C.
      • Chakraborty A.
      • Chou W.M.
      • et al.
      Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
      The lifespan of individual cccDNA molecules is not well understood and in the setting of an antiviral immune response may be shorter than the life of a hepatocyte.
      • Bockmann J.H.
      • Stadler D.
      • Xia Y.
      • et al.
      Comparative analysis of the antiviral effects mediated by type I and III interferons in hepatitis B virus infected hepatocytes.
      In cell culture systems, cccDNA has been estimated to have a half-life of approximately 40 days,
      • Ko C.
      • Chakraborty A.
      • Chou W.M.
      • et al.
      Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
      • Zhu Y.
      • Yamamoto T.
      • Cullen J.
      • et al.
      Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.
      whereas human studies evaluating cccDNA turnover have suggested that populations of cccDNA can convert from mutant to wild type (or via versa) in as little as 12 weeks.

      Huang Q, Zhou B, Zong Y, et al. Rapid turnover of cccDNA in chronic hepatitis B patients who have failed nucleoside treatment due to emerging resistance. The Liver Meeting. Washington, DC, October 20–24, 2017.

      However, cccDNA may persist longer in a residual pool of quiescent nondividing hepatocytes.
      • Yang H.C.
      • Kao J.H.
      Persistence of hepatitis B virus covalently closed circular DNA in hepatocytes: molecular mechanisms and clinical significance.
      cccDNA is not known to tether to the nuclear spindle, and most cccDNA is thought to be lost during hepatocyte expansion, as occurs, for example, following an immune response and clearance of infected cells. Importantly, HBV is not a cytopathic virus; thus, in the absence of an adequate antiviral immune response, there is not thought to be a virus-derived trigger to drive hepatocyte loss or proliferation.
      Possible targets for therapeutic intervention include HBV binding and absorption, capsid dissociation, cccDNA formation, gene expression, protein synthesis, capsid assembly, viral DNA synthesis, capsid envelopment, and virion release (Fig. 1). Most of these targets are being addressed by ongoing therapeutic research programs. In addition, because attenuation of the HBV-specific immune response is a key feature of chronic HBV infection, multiple approaches are being explored to restore immune responsiveness to allow immune-mediated clearance of HBV-infected hepatocytes.
      Figure thumbnail gr1
      Fig. 1HBV replication cycle and therapeutic targets. The steps of HBV replication are shown, beginning with viral entry into a hepatocyte. The inset table indicates active therapeutic research targets associated with each step.

       Goal of New Therapies

      The ultimate goal of ongoing research for novel HBV therapeutics is eradication of the cccDNA viral reservoir. Because measurement of cccDNA itself is challenging, requiring a biopsy and sophisticated assays that have not yet been standardized, surrogates for cccDNA loss or inactivation are required. These surrogates must include a sustained viral response (SVR), lack of viral rebound with persistent normal liver function and lack of inflammation, after a finite course of therapy. In addition to loss of detectable serum HBV DNA, additional biomarker changes expected to coincide with such improved off-treatment responses include a loss of serum HBeAg and either loss or a decline to a stable low level of detectable serum HBsAg.
      • Vigano M.
      • Lampertico P.
      Clinical implications of HBsAg quantification in patients with chronic hepatitis B.
      Loss of HBsAg in particular, although uncommon with current therapies, has been associated with sustained posttreatment virologic responses and is generally considered a clinical marker of a functional cure, indicating that treatment is no longer required.
      • Vigano M.
      • Lampertico P.
      Clinical implications of HBsAg quantification in patients with chronic hepatitis B.
      Although HBsAg clearance is accepted as a desirable endpoint, its achievement has been somewhat confounded by the discovery that, at least in chimpanzees, circulating HBsAg may arise from HBV integrants, which, although unable to produce complete virus, may produce a significant fraction of the total circulating HBsAg.
      • Wooddell C.I.
      • Yuen M.F.
      • Chan H.L.
      • et al.
      RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.
      Unfortunately, even in the rare instances in which HBsAg loss has been achieved, reactivation in the setting of immunosuppression is possible. The exact mechanisms by which immune control and escape occur are only partially understood, suggesting that the understanding of even such “functional cures” is still evolving.
      • Loomba R.
      • Liang T.J.
      Hepatitis B reactivation associated with immune suppressive and biological modifier therapies: current concepts, management strategies, and future directions.
      Given the challenges of defining what constitutes a “cure” for CHB (functional or otherwise) with today’s technology, the author proposes that a more appropriate definition of the clinical goals for current studies may be similar to those used as hepatitis C therapeutics were being developed. An initial objective should be “sustained viral response” as measured by lack of viral DNA or antigen rebound for a fixed period off treatment. Subsequently, “cure” could be declared in individuals in whom SVR is maintained over a more extended time frame.

       Current Options

      Current options for CHB therapy include nucleos(t)ide analogues and interferons. In brief, nearly all patients respond during treatment with nucleos(t)ide analogues, but beneficial responses are rarely sustained after treatment. Interferons, in contrast, may provide sustained responses more frequently, but only in a limited subset of patients defined primarily by HBV genotype and disease characteristics.
      • Flink H.J.
      • van Zonneveld M.
      • Hansen B.E.
      • et al.
      Treatment with peg-interferon alpha-2b for HBeAg-positive chronic hepatitis B: HBsAg loss is associated with HBV genotype.
      • Sonneveld M.J.
      • Rijckborst V.
      • Boucher C.A.
      • et al.
      Prediction of sustained response to peginterferon alfa-2b for hepatitis B e antigen-positive chronic hepatitis B using on-treatment hepatitis B surface antigen decline.
      • Tseng T.C.
      • Yu M.L.
      • Liu C.J.
      • et al.
      Effect of host and viral factors on hepatitis B e antigen-positive chronic hepatitis B patients receiving pegylated interferon-alpha-2a therapy.
      Although neither of these therapies, alone or in combination, has dramatically increased cure rates in patients with CHB, it is highly likely that a successful curative regimen will entail combinations with either or both of these modalities.

       Interferons

      In 1991, interferon alfa-2b became the first treatment approved for CHB therapy; pegylated interferon alfa-2a was approved in 2005. Interferons are typically administered by subcutaneous injection for up to 12 months and can elicit durable posttreatment suppression of HBV replication and antigenemia.
      • Arends P.
      • Rijckborst V.
      • Zondervan P.E.
      • et al.
      Loss of intrahepatic HBsAg expression predicts sustained response to peginterferon and is reflected by pronounced serum HBsAg decline.
      Tolerability can be poor, however, and clinically significant response rates are generally achievable by only a small subset of patients.

       Nucleos(t)ide Analogues

      The most commonly used therapies for CHB at present, nucleos(t)ide HBV pol/RT inhibitors, profoundly inhibit HBV DNA synthesis, frequently reducing serum HBV DNA to levels at the limit of quantification. This response is associated with improvement of hepatic fibrosis and reduced risk of hepatocellular carcinoma. All agents in this class have similar modes of action. Lamivudine, approved in the United States in 1998, was the first antiviral nucleos(t)ide analogue available for treatment of CHB. Subsequently, adefovir (2002), entecavir (2005), telbivudine (2006), tenofovir disoproxil fumarate (2008), and tenofovir alafenamide (2016) were approved. Clevudine and besifovir dipivoxil maleate are approved in South Korea but not in the United States. Agents in this class exhibit some differences with respect to potency, safety, and resistance profiles. The most important difference is the superior barrier to resistance exhibited by tenofovir and entecavir, as demonstrated by negligible rates of virologic failure even after years of therapy.
      • Chang T.T.
      • Lai C.L.
      • Kew Yoon S.
      • et al.
      Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B.
      • Liu Y.
      • Corsa A.C.
      • Buti M.
      • et al.
      No detectable resistance to tenofovir disoproxil fumarate in HBeAg+ and HBeAg- patients with chronic hepatitis B after 8 years of treatment.
      Tenofovir alafenamide elicits virologic responses similar to those associated with tenofovir disoproxil fumarate, but with reduced risk of bone and renal toxicities.
      • Buti M.
      • Gane E.
      • Seto W.K.
      • et al.
      Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of patients with HBeAg-negative chronic hepatitis B virus infection: a randomised, double-blind, phase 3, non-inferiority trial.
      • Chan H.L.
      • Fung S.
      • Seto W.K.
      • et al.
      Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of HBeAg-positive chronic hepatitis B virus infection: a randomised, double-blind, phase 3, non-inferiority trial.

      Seto WK, Buti M, Izumi N, et al. Bone and renal safety are improved in chronic HBV patients 1 year after switching to tenofovir alafenamide from tenofovir disoproxil fumarate. The Liver Meet. San Francisco, CA, November 9–13, 2018.

      Despite their success in persistently inhibiting HBV pol/RT, because cccDNA formation from rcDNA is catalyzed primarily by cellular polymerases, nucleos(t)ide analogues have minimal effects on cccDNA establishment and affect HBV gene expression only indirectly.
      • Moraleda G.
      • Saputelli J.
      • Aldrich C.E.
      • et al.
      Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.
      Over the last several years, several lines of evidence have also suggested that nucleos(t)ide analogues alone are unable to fully inhibit all HBV replication. For example, analysis of liver biopsies has shown that HBV replication often persists at low levels even after years of nucleos(t)ide analogue therapy.
      • Boyd A.
      • Lacombe K.
      • Lavocat F.
      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      As long as low-level replication persists, new hepatocytes can be reinfected continuously, and it is unlikely that nucleos(t)ide analogues alone will cure most individuals; clinically, this is apparent from the slow HBsAg declines and low cure rates seen on current standards of care.
      • Alidjinou E.K.
      • Michel C.
      • Canva V.
      • et al.
      Very slow decline of hepatitis B virus surface antigen and core related antigen in chronic hepatitis B patients successfully treated with nucleos(t)ide analogues.
      As a result, for most patients, lengthy if not lifelong treatment is likely to be needed. In those few patients in whom both serum antigens and HBV DNA do become undetectable, a sustained posttreatment virologic response is often achieved if therapy is stopped, supporting HBsAg clearance as a possible biomarker of functional cure.
      • Vigano M.
      • Lampertico P.
      Clinical implications of HBsAg quantification in patients with chronic hepatitis B.
      • Chi H.
      • Wong D.
      • Peng J.
      • et al.
      Durability of response after hepatitis B surface antigen seroclearance during nucleos(t)ide analogue treatment in a multiethnic cohort of chronic hepatitis B patients: results after treatment cessation.
      • Van Hees S.
      • Chi H.
      • Hansen B.
      • et al.
      Sustained off-treatment viral control is associated with high hepatitis B surface antigen seroclearance rates in Caucasian patients with nucleos(t)ide analogue induced HBeAg seroconversion.
      Combination therapy with a nucleos(t)ide analogue and pegylated interferon has been explored with generally disappointing results.
      • Arends J.E.
      • Lieveld F.I.
      • Ahmad S.
      • et al.
      New viral and immunological targets for hepatitis B treatment and cure: a review.
      • Su T.H.
      • Liu C.J.
      Combination therapy for chronic hepatitis B: current updates and perspectives.
      Although a few studies have reported higher rates of sustained posttreatment responses with peginterferon combined with entecavir or tenofovir versus interferon alone,
      • Brouwer W.P.
      • Xie Q.
      • Sonneveld M.J.
      • et al.
      Adding pegylated interferon to entecavir for hepatitis B e antigen-positive chronic hepatitis B: a multicenter randomized trial (ARES study).
      • Marcellin P.
      • Ahn S.H.
      • Ma X.
      • et al.
      Combination of tenofovir disoproxil fumarate and peginterferon alpha-2a increases loss of hepatitis B surface antigen in patients with chronic hepatitis B.
      • Ahn S.H.
      • Marcellin P.
      • Ma X.
      • et al.
      Hepatitis B surface antigen loss with tenofovir disoproxil fumarate plus peginterferon alfa-2a: week 120 analysis.
      response rates are typically low and the population likely to benefit appears similar to that with interferon alone.
      • Marcellin P.
      • Ahn S.H.
      • Chuang W.L.
      • et al.
      Predictors of response to tenofovir disoproxil fumarate plus peginterferon alfa-2a combination therapy for chronic hepatitis B.
      Several Asian studies have reported no incremental benefit from combination therapies.
      • Chi H.
      • Hansen B.E.
      • Guo S.
      • et al.
      Pegylated interferon alfa-2b add-on treatment in hepatitis B virus envelope antigen-positive chronic hepatitis B patients treated with nucleos(t)ide analogue: a randomized, controlled trial (PEGON).
      • Hsu C.W.
      • Su W.W.
      • Lee C.M.
      • et al.
      Phase IV randomized clinical study: peginterferon alfa-2a with adefovir or entecavir pre-therapy for HBeAg-positive chronic hepatitis B.
      • Jun D.W.
      • Ahn S.B.
      • Kim T.Y.
      • et al.
      Efficacy of pegylated interferon monotherapy versus sequential therapy of entecavir and pegylated interferon in hepatitis B e antigen-positive hepatitis B patients: a randomized, multicenter, phase IIIb open-label study (POTENT study).
      • Xie Q.
      • Zhou H.
      • Bai X.
      • et al.
      A randomized, open-label clinical study of combined pegylated interferon alfa-2a (40KD) and entecavir treatment for hepatitis B “e” antigen-positive chronic hepatitis B.

       Novel Therapies

      Novel therapies in clinical development include both direct-acting antivirals (DAAs), which target viral proteins or viral RNAs, and host-directed antivirals, such as entry inhibitors and immunomodulators (Table 1). Additional approaches, such as direct cccDNA targeting, epigenetic modifiers, and RNAse H inhibition, remain preclinical but are anticipated to initiate clinical evaluation over the next few years.
      Table 1Therapeutics in clinical development for chronic hepatitis B
      Drug ClassDrugCompanyPhase 1Phase 2Phase 3
      DAAs
       Core protein inhibitorsAB-506Arbutus Biopharma
      ABI-H0731Assembly Biosciences
      ABI-H2158Assembly Biosciences
      EDP-514Enanta Pharmaceuticals
      JNJ-6379Johnson & Johnson
      JNJ-0440Johnson & Johnson
      RO7049389Roche
       siRNA, antisense RNAAB-729Arbutus Biopharma
      DCR-HBVSDicerna Pharmaceuticals
      GSK/IONIS-HBV-LRxIonis/GlaxoSmithKline
      IONIS-HBVRxIonis/GlaxoSmithKline
      JNJ-3989 (ARO-HBV)Johnson & Johnson
      RO7062931Roche
      Vir-2218 (ALN-HBV02)Vir Biotechnology/Alnylam
       pol/RT inhibitorTenofovir exalidexContraVir Pharmaceuticals
       HBsAg secretion inhibitorsREP-2139Replicor
      REP-2165Replicor
      Indirect-acting antivirals and immunotherapeutics
       HBV entry inhibitorBulevirtideHepatera Ltd
       TLR-7 agonistsAL-034Johnson & Johnson/Alios
      RG-7854Roche
      RO7020531Roche
       TLR-8 agonistGS-9688Gilead Sciences
       Therapeutic vaccinesAIC-649AiCuris
      INO-1800Inovio Pharmaceuticals
      TG1050Transgene
       RIG-I and NOD2 agonistInarigivirSpring Bank
       Apoptosis inducerAPG-1387Ascentage Pharma
       FXR agonistEYP-001Enyo Pharma
      Abbreviations: NOD2, nucleotide-binding oligomerization domain-containing protein 2; RIG-I, retinoic acid-inducible gene-I.

      Therapies in clinical development

      As discussed, inhibition of the HBV pol/RT by nucleos(t)ide analogues is of limited efficacy owing to their primary mode of action, which has little impact on either cccDNA establishment or viral gene expression. They have repeatedly been shown to incompletely suppress intrahepatic viral replication,
      • Boyd A.
      • Lacombe K.
      • Lavocat F.
      • et al.
      Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.
      • Marcellin P.
      • Buti M.
      • Krastev Z.
      • et al.
      Kinetics of hepatitis B surface antigen loss in patients with HBeAg-positive chronic hepatitis B treated with tenofovir disoproxil fumarate.

      Burdette D. Evidence for the presence of infectious virus in the serum from chronic hepatitis B patients suppressed on nucleos(t)ide therapy with detectable but not quantifiable HBV DNA. The Int Liver Congress. Vienna, Austria, April 10–14, 2019.

      resulting in rapid recurrence of viral replication if treatment is stopped. Over the past 5 to 10 years, other aspects of the HBV replication cycle have been explored with the aim of identifying agents that could address these limitations and achieve antiviral effects that persist after treatment.

       Direct-Acting Antivirals

      Novel DAAs are being developed with the rationale that hepatocytes have a finite lifespan; thus, if new (uninfected) hepatocytes can be protected from infection by a potent antiviral regimen, then the duration of the infection cannot exceed the lifespan of the hepatocytes and may be shorter if cccDNA loss is more rapid than hepatocyte loss.
      • Ko C.
      • Chakraborty A.
      • Chou W.M.
      • et al.
      Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.
      • Xia Y.
      • Stadler D.
      • Lucifora J.
      • et al.
      Interferon-gamma and tumor necrosis factor-alpha produced by T cells reduce the HBV persistence form, cccDNA, without cytolysis.
      • Guidotti L.G.
      • Ishikawa T.
      • Hobbs M.V.
      • et al.
      Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes.
      If current standard-of-care nucleos(t)ide therapies are, in fact, achieving low cure rates as a result of ongoing intrahepatic HBV replication (ie, primary antiviral failure), then combining a nucleos(t)ide analogue with a DAA for a sufficient length of time may be all that is required to achieve a cure for CHB. DAAs in development include entry inhibitors, RNA inhibitors, and core protein inhibitors.

       Entry Inhibitors

      Blockade of HBV entry into hepatocytes, thus preventing the earliest step of infection, has been explored concurrent with identification of NTCP as the HBV receptor.
      • Yan H.
      • Zhong G.
      • Xu G.
      • et al.
      Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
      Bulevirtide (myrcludex B; Hepatera Ltd), a specific NTCP inhibitor, blocks attachment of viral pre-S1 via high-affinity binding to NTCP.
      • Urban S.
      • Bartenschlager R.
      • Kubitz R.
      • et al.
      Strategies to inhibit entry of HBV and HDV into hepatocytes.
      Current clinical studies of bulevirtide are focused on patients with hepatitis delta virus (HDV) coinfection. Because infectivity of both HBV and HDV depends on the presence of HBsAg in the viral envelope, these studies will assess virologic responses of both HDV and HBV. Phase 2 data showed a marked reduction of HDV RNA after 24 weeks of bulevirtide combined with tenofovir,

      Wedemeyer H, Bogomolov P, Blank A, et al. Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with tenofovir in patients with HBV/HDV coinfection. The Int Liver Congress. Paris, France, April 11–15, 2018.

      and after 48 weeks of bulevirtide with or without peginterferon-alfa.

      Wedemeyer H, Schoneweis K, Bogomolov P, et al. Interim results of a multicenter, open-label phase 2 clinical trial (MYR203) to assess safety and efficacy of Myrcludex B in combination with PEG-IFNα in patients with chronic HBV/HDV co-infection. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      In the latter study, the HBsAg decline was greater with bulevirtide in combination with peginterferon-alfa. Planned phase 3 studies will explore extended therapy with bulevirtide as monotherapy or in combination with peginterferon-alfa in patients with HBV/HDV coinfection, focusing on HDV clearance as the primary objective. Although entry blockers would not be expected to directly interfere with HBV cccDNA formation, entry inhibition with bulevirtide would be anticipated to protect still uninfected cells and thus may contribute to HBV therapy either alone or in combination with other modalities.

       RNA Interference

      RNA interference (RNAi) is a natural process by which a small interfering RNA (siRNA) duplex directs sequence-specific posttranscriptional silencing by binding to complementary mRNA, triggering its elimination.
      • Chen Y.
      • Cheng G.
      • Mahato R.I.
      RNAi for treating hepatitis B viral infection.
      • Flisiak R.
      • Jaroszewicz J.
      • Lucejko M.
      siRNA drug development against hepatitis B virus infection.
      Because the HBV genome is compact with multiple overlapping reading frames, targeting viral transcripts with siRNA has emerged as an attractive approach anticipated to reduce expression of multiple viral antigens. Of particular interest has been the implication of reducing HBsAg, which has been proposed to contribute to specific impairment of an effective anti-HBV immune response.
      Various approaches have been taken to safely enhance delivery of the siRNA moiety and to target the HBV genome within hepatocytes. Although most of these programs remain preclinical, several have entered human studies with mixed results. An early siRNA against HBV, ARC-520 (Arrowhead Pharmaceuticals), was composed of 2 siRNAs conjugated to cholesterol and administered with a polymer-based system containing N-acetyl galactosamine; together these conjugates enhance delivery to the hepatocyte cytoplasm.
      • Schluep T.
      • Lickliter J.
      • Hamilton J.
      • et al.
      Safety, tolerability, and pharmacokinetics of ARC-520 injection, an RNA interference-based therapeutic for the treatment of chronic hepatitis B virus infection, in healthy volunteers.
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      • et al.
      Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.
      Monthly injections of ARC-520 in combination with standard oral entecavir achieved multilog reductions of serum HBsAg concentrations in HBeAg-positive patients that persisted for months after discontinuing ARC-520; lesser (<1 log) reductions of HBsAg were reported in HBeAg-negative patients.

      Yuen M-F, Liu K, Given B, et al. RNA interference therapy with ARC-520 injection results in long term off-therapy antigen reductions in treatment naïve, HBeAg positive and negative patients with chronic HBV. The Int Liver Congress. Paris, France, April 11–15, 2018.

      These results helped demonstrate the significant percentage of circulating HBsAg that could be derived from integrated HBV sequences in HBeAg-negative patients. Subsequently, 3 monthly subcutaneous injections of a follow-on product, JNJ-3989 (formerly ARO-HBV; Johnson & Johnson/Arrowhead), which targets a different region of the HBV genome, also elicited dose-related reductions of serum HBsAg of 1.3 to 3.8 log10 IU/mL, as well as reductions of HBV DNA, HBV RNA, HBeAg, and hepatitis B core-related antigen.

      Gane EJ, Locarnini S, Lim TH, et al. First results with RNA interference (RNAi) in chronic hepatitis B (CHB) using ARO-HBV. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Gane EJ, Locarnini S, Lim TH, et al. RNA interference (RNAi) in chronic hepatitis B (CHB): data from phase 2 study with JNJ-3989. 28th annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

      Viral antigen responses were similar with or without concomitant nucleos(t)ide analogue therapy and in both HBeAg-positive and HBeAg-negative patients, in contrast to previous results with ARC-520.

      Yuen M-F, Liu K, Given B, et al. RNA interference therapy with ARC-520 injection results in long term off-therapy antigen reductions in treatment naïve, HBeAg positive and negative patients with chronic HBV. The Int Liver Congress. Paris, France, April 11–15, 2018.

      Yuen M-F, Chan HL-Y, Liu K, et al. Differential reductions in viral antigens expressed from cccDNA vs integrated DNA in treatment naive HBeAg positive and negative patients with chronic HBV after RNA interference therapy with ARC-520. The International Liver Congress. Barcelona, Spain, April 13–17, 2016.

      AB-729 (Arbutus) is a subcutaneously delivered N-acetylgalactosamine-conjugated siRNA moiety that demonstrated significant suppression of HBsAg in mouse models of HBV infection.

      Lee ACH. Durable inhibition of hepatitis B virus replication and antigenemia using subcutaneously administered siRNA agent AB-729 in preclinical models. The International Liver Congress. Paris, France, April 11–15, 2018.

      Initial clinical evaluation is planned for 2019. Clinical development of ARB-1467, Arbutus’ initial product in this category, has been discontinued.

       Core Protein Inhibitors

      The HBV core protein plays several essential roles in the HBV life cycle, including capsid formation.
      • Mak L.Y.
      • Wong D.K.
      • Seto W.K.
      • et al.
      Hepatitis B core protein as a therapeutic target.
      • Selzer L.
      • Zlotnick A.
      Assembly and release of hepatitis B virus.
      • Zlotnick A.
      • Venkatakrishnan B.
      • Tan Z.
      • et al.
      Core protein: a pleiotropic keystone in the HBV lifecycle.
      • Tan Z.
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      • Unchwaniwala N.
      • et al.
      The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription.

      Huang Q, Mercier A, Zhou Y, et al. Preclinical characterization of potent core protein assembly modifiers for the treatment of chronic hepatitis B. The International Liver Congress. Barcelona, Spain, April 13–17, 2016.

      Huang Q, Turner WW, Haydar S, et al. Preclinical profile of potent second generation CpAMs capable of inhibiting the generation of HBsAg, HBeAg, pgRNA and cccDNA in HBV-infected cells. The Liver Meeting. Washington, DC, October 20–24, 2017.

      Rijnbrand R. Preclinical antiviral drug combination studies utilizing novel orally bioavailable agents for chronic hepatitis B infection: AB-506, a next generation HBV capsid inhibitor, and AB-452, an HBV RNA destabilizer. The International Liver Congress. Paris, France, April 11–15, 2018.

      Zhang Z, Liang B, Jin Q, et al. A novel HBV capsid formation inhibitor that additively inhibits virus replication in combination with tenofovir or IFN-α. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Zhou X, Zhou Y, Tian X, et al. In vitro and in vivo antiviral characterization of RO7049389, a novel small molecule capsid assembly modulator, for the treatment of chronic hepatitis B. The International Liver Congress. Paris, France, April 11–15, 2018.

      • Yang L.
      • Liu F.
      • Tong X.
      • et al.
      Treatment of chronic hepatitis B virus infection using small molecule modulators of nucleocapsid assembly: recent advances and perspectives.
      The kinetics of core protein assembly are critical for functional capsid formation, encapsidation of pgRNA, and formation of infectious virions. The HBV capsid is also essential for nuclear importation of HBV rcDNA through a regulated interaction with nuclear pore proteins, allowing replenishment of nuclear cccDNA pools from either newly formed capsids or incoming virus.
      • Guo F.
      • Zhao Q.
      • Sheraz M.
      • et al.
      HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.
      Nuclear forms of the HBV core protein have been implicated in modulating the expression of viral and host genes and contributing to regulated splicing and nuclear export of HBV RNAs, and to cccDNA function.
      • Belloni L.
      • Allweiss L.
      • Guerrieri F.
      • et al.
      IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome.
      • Diab A.
      • Foca A.
      • Zoulim F.
      • et al.
      The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: implications for the development of HBc-targeting antivirals.
      These findings suggest that allosteric modulation of the core protein would allow targeting of multiple aspects of the viral life cycle and predict that HBV core protein inhibitors (also known as core inhibitors, or core protein allosteric modulators) may be potent antivirals. Several classes of core inhibitors have been described across multiple chemotypes
      • Zhang X.
      • Cheng J.
      • Ma J.
      • et al.
      Discovery of novel hepatitis B virus nucleocapsid assembly inhibitors.
      ; all are thought to bind to the same pocket in the dimer-dimer interface, nucleating inappropriate oligomerization and rendering nascent capsids unable to encapsidate viral RNA. Subtle differences in core oligomer morphologies following addition of core inhibitors in vitro have been described, ranging from empty capsids to “cracked” capsids with potential changes in cellular localization following core protein aggregation.
      • Schlicksup C.J.
      • Wang J.C.
      • Francis S.
      • et al.
      Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.
      How these may relate to clinical outcomes remains to be demonstrated. Importantly, all core inhibitors in clinical development exhibit a common property: in addition to potent inhibition of new rcDNA formation, all inhibit viral RNA packaging, and at higher doses, may inhibit the establishment of cccDNA. In contrast, nucleos(t)ide analogues inhibit neither of these processes to a significant degree.
      Preclinically, several core inhibitors have shown attractive profiles as once-daily oral therapeutics, with potential for additive to synergistic activity in combination with nucleos(t)ide analogues.

      Bassit L, Cox B, Ono SK, et al. Novel and potent HBV capsid modulator reduces HBeAg and cccDNA in core-site directed T109I mutant in HepNTCP cells. The International Liver Congress. Paris, France, April 11–15, 2018.

      Berke JM, Dehertogh P, Vergauwen K, et al. In vitro antiviral activity and mode of action of JNJ-64530440, a novel potent hepatitis B virus capsid assembly modulator in clinical development. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Berke JM, Verbinnen T, Tan Y, et al. The HBV capsid assembly modulator JNJ-379 is a potent inhibitor of viral replication across full length genotype A-H clinical isolates in vitro. The Liver Meeting. Washington, DC, October 20–24, 2017.

      Several core inhibitors are currently in clinical evaluation.

      Eley T, Caamano S, Denning J, et al. Single dose safety, tolerability and pharmacokinetics of AB-423 in healthy volunteers from the ongoing single and multiple ascending dose study AB-423-001. The Liver Meeting. Washington, DC, October 20–24, 2017.

      Gane E, Liu A, Yuen MF, et al. RO7049389, a core protein allosteric modulator, demonstrates robust anti-HBV activity in chronic hepatitis B patients and is safe and well tolerated. The International Liver Congress. Paris, France, April 11–15, 2018.

      Kakuda TN, Yogaratnam JZ, Gane EJ, et al. Single-dose pharmacokinetics, safety and tolerability of JNJ-64530440, a novel hepatitis B virus capsid assembly modulator, in healthy volunteers. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Yuen MF, Agarwal K, Gane EJ, et al. Interim safety, tolerability, pharmacokinetics, and antiviral activity of ABI-H0731, a novel core protein allosteric modifier (CpAM), in healthy volunteers and non-cirrhotic viremic subjects with chronic hepatitis B. The International Liver Congress. Paris, France, April 11–15, 2018.

      Yuen MF, Agarwal K, Gane EJ, et al. Final results of a phase 1B 28-day study of ABI-H0731, a novel core inhibitor, in non-cirrhotic viremic subjects with chronic HBV. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      • Yuen M.F.
      • Gane E.J.
      • Kim D.J.
      • et al.
      Antiviral activity, safety, and pharmacokinetics of capsid assembly modulator NVR 3-778 in patients with chronic HBV infection.

      Zoulim F, Yogaratnam JZ, Vandenbossche JJ, et al. Safety, tolerability, pharmacokinetics, and antiviral activity of JNJ-56136379, a novel HBV capsid assembly modulator, in non-cirrhotic, treatment-naïve patients with chronic hepatitis B. The Liver Meeting. Washington, DC, October 20–24, 2017.

      Zoulim F, Yogaratnam JZ, Vandenbossche JJ, et al. Safety, pharmacokinetics and antiviral activity of novel HBV capsid assembly modulator, JNJ-56136379, in patients with chronic hepatitis B. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Early data from phase 1b monotherapy studies suggest that this novel class has the potential to be at least as potent as nucleos(t)ide analogue monotherapy, with single-agent HBV DNA declines of up to 4 log10 IU/mL, as well as 2 to 3 log10 declines in viral RNA over ≤28 days.

      Yuen MF, Agarwal K, Gane EJ, et al. Interim safety, tolerability, pharmacokinetics, and antiviral activity of ABI-H0731, a novel core protein allosteric modifier (CpAM), in healthy volunteers and non-cirrhotic viremic subjects with chronic hepatitis B. The International Liver Congress. Paris, France, April 11–15, 2018.

      Yuen MF, Agarwal K, Gane EJ, et al. Final results of a phase 1B 28-day study of ABI-H0731, a novel core inhibitor, in non-cirrhotic viremic subjects with chronic HBV. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Zoulim F, Yogaratnam JZ, Vandenbossche JJ, et al. Safety, pharmacokinetics and antiviral activity of novel HBV capsid assembly modulator, JNJ-56136379, in patients with chronic hepatitis B. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Yogaratnam JZ, Zoulim F, Vandenbossche JJ, et al. Safety and antiviral activity of a novel hepatitis B virus capsid assembly modulator, JNJ-56136379, in Asian and non-Asian patients with chronic hepatitis B. 28th Annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

      Zoulim F, Yogaratnam JZ, Vandenbossche JJ, et al. Safety, pharmacokinetics, and antiviral activity of a novel hepatitis B virus capsid assembly modulator, JNJ-56136379, in patients with chronic hepatitis B. 28th Annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

      Early data from phase 2 evaluations of core inhibitors are anticipated in 2019, including combinations with other therapeutic modalities. If preclinical data with these combinations, suggesting additive to synergistic interactions,

      Rijnbrand R. Preclinical antiviral drug combination studies utilizing novel orally bioavailable agents for chronic hepatitis B infection: AB-506, a next generation HBV capsid inhibitor, and AB-452, an HBV RNA destabilizer. The International Liver Congress. Paris, France, April 11–15, 2018.

      Zhang Z, Liang B, Jin Q, et al. A novel HBV capsid formation inhibitor that additively inhibits virus replication in combination with tenofovir or IFN-α. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Dai L, Yu Y, Zhou X, et al. Combination treatment of a TLR7 agonist RO7020531 and a core protein allosteric modulator RO7049389 achieved sustainable viral load suppression and HBsAg loss in an AAV-HBV mouse model. The International Liver Congress. Paris, France, April 11–15, 2018.

      • Klumpp K.
      • Shimada T.
      • Allweiss L.
      • et al.
      Efficacy of NVR 3-778, alone and in combination with pegylated interferon, vs entecavir in uPA/SCID mice with humanized livers and HBV infection.
      are reflected by anticipated enhanced declines in viral load, it will be extremely interesting to assess whether these effects are followed by viral antigen reductions or loss, which would be predicted if an enhanced antiviral effect reduces cccDNA persistence.

       Novel Viral Polymerase Inhibitors

      Besifovir dipivoxil maleate has recently been approved in Korea, based on phase 3 data indicating noninferior HBV DNA suppression versus tenofovir disoproxil fumarate, with significantly reduced bone and renal toxicities at 48 weeks.
      • Ahn S.H.
      • Kim W.
      • Jung Y.K.
      • et al.
      Efficacy and safety of besifovir dipivoxil maleate compared with tenofovir disoproxil fumarate in treatment of chronic hepatitis B virus infection.
      However, further clinical evaluation appears to be limited to studies in Korea, suggesting that broader registration efforts may not be forthcoming. A lipid-conjugated, liver-targeted prodrug of tenofovir (tenofovir exalidex; ContraVir) is in early clinical development, with the primary objective of improving safety over current tenofovir disoproxil fumarate formulations.

      Tanwandee T, Chatsiricharoenkul S, Tangkijvanich P, et al. Pharmacokinetics, safety and tolerability of tenofovir exalidex, a novel prodrug of tenofovir, administered as ascending multiple doses to healthy volunteers and HBV-infected subjects. The International Liver Congress. Amsterdam, The Netherlands, April 19–23, 2017.

       Immunotherapeutic Strategies

      From an immunologic perspective, it has been shown that a persistent, HBV-specific immunologic dysfunction exists in CHB, with both a paucity and a functional impairment of HBV-specific T cells.
      • Bertoletti A.
      • Maini M.K.
      • Ferrari C.
      The host-pathogen interaction during HBV infection: immunological controversies.
      • Rehermann B.
      • Thimme R.
      Insights from antiviral therapy into immune responses to hepatitis B and C virus infection.
      Although the nature of this dysfunction is only partially understood, multiple potential immunotherapeutic approaches have been suggested as a means to achieve a cure by restoring immune competence against HBV and HBV-infected hepatocytes.
      • Bertoletti A.
      • Le Bert N.
      Immunotherapy for chronic hepatitis B virus infection.
      • Salimzadeh L.
      • Le Bert N.
      • Dutertre C.A.
      • et al.
      PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.
      • Lim S.G.
      • Agcaoili J.
      • De Souza N.N.A.
      • et al.
      Therapeutic vaccination for chronic hepatitis B: a systematic review and meta-analysis.
      • Maini M.K.
      • Gehring A.J.
      The role of innate immunity in the immunopathology and treatment of HBV infection.
      • Gehring A.J.
      • Protzer U.
      Targeting innate and adaptive immune responses to cure chronic HBV infection.
      A thesis underlying immunotherapeutic approaches is that HBV is a disease that can be fully suppressed and functionally cured in the context of most (adult) acute infections. If the deficit that allows CHB persistence could be resolved, then immune restoration may allow a similar clinical cure for CHB. Immunotherapeutics in clinical development include toll-like receptor (TLR)7 AND TLR8 agonists, checkpoint inhibitors (eg, anti-PD-1), an RIG-I agonist, and therapeutic vaccines.

       Toll-like Receptor Agonists

      TLRs are intracellular pathogen-sensing receptors that, when triggered, can arm the immune response to produce antiviral cytokines, such as interferons alfa and gamma, and induce activation of both natural killer and T cells.
      • Ma Z.
      • Cao Q.
      • Xiong Y.
      • et al.
      Interaction between hepatitis B virus and toll-like receptors: current status and potential therapeutic use for chronic hepatitis B.
      • Boni C.
      • Vecchi A.
      • Rossi M.
      • et al.
      TLR7 agonist increases responses of hepatitis B virus-specific T cells and natural killer cells in patients with chronic hepatitis B treated with nucleos(t)ide analogues.
      In animal models, both TLR7 and TLR8 have shown an impressive ability to cure woodchucks of woodchuck hepatitis virus infection, although tolerability has arisen as a potential concern.
      • Menne S.
      • Tumas D.B.
      • Liu K.H.
      • et al.
      Sustained efficacy and seroconversion with the toll-like receptor 7 agonist GS-9620 in the woodchuck model of chronic hepatitis B.
      • Lanford R.E.
      • Guerra B.
      • Chavez D.
      • et al.
      GS-9620, An oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees.
      The ability to deliver TLR agonists such as Gilead TLR7 agonist vesatolimod (GS-9620)
      • Agarwal K.
      • Ahn S.H.
      • Elkhashab M.
      • et al.
      Safety and efficacy of vesatolimod (GS-9620) in patients with chronic hepatitis B who are not currently on antiviral treatment.
      and TLR8 agonist (GS-9688)

      Gane E, Kim HJ, Visvanathan K, et al. Safety, pharmacokinetics, and pharmacodynamics of oral TLR8 agonist GS-9688 in patients with chronic hepatitis B: a randomized, placebo-controlled, double-blind phase 1b study. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      orally has led to the hope that a “presystemic” antiviral immune response that was limited to the portal circulation might be generated, potentially providing the benefits seen with injected interferons without systemic side effects. Although early clinical results with vesatolimod in patients with CHB were disappointing,
      • Agarwal K.
      • Ahn S.H.
      • Elkhashab M.
      • et al.
      Safety and efficacy of vesatolimod (GS-9620) in patients with chronic hepatitis B who are not currently on antiviral treatment.
      GS-9688 as well as TLR7 agonists from Roche (RO7020531) and Johnson & Johnson (JNJ-4964) are still advancing in clinical trials and have been shown to successfully stimulate immune responses in both healthy volunteers and patients with CHB.

      Gane E, Kim HJ, Visvanathan K, et al. Safety, pharmacokinetics, and pharmacodynamics of oral TLR8 agonist GS-9688 in patients with chronic hepatitis B: a randomized, placebo-controlled, double-blind phase 1b study. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Gane E, Agarwal K, Balabanska R, et al. TLR7 agonist RO7020531 triggers immune activation after multiple doses in chronic hepatitis B patients. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Gane E, Kim HJ, Visvanathan K, et al. Safety, pharmacokinetics, and pharmacodynamics of oral TLR8 agonist GS-9688 in patients with chronic hepatitis B: a randomized, placebo-controlled, double-blind phase 1b study. 28th Annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

      Luk A, Grippo JF, Folitar I, et al. A single and multiple ascending dose study of toll-like receptor 7 (TLR7) agonist (RO7020531) in Chinese healthy subjects. 28th Annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

       Checkpoint Blockers

      The presence of an “exhausted” CD8+ T-cell phenotype in patients with CHB has spurred exploration of PD1-PDL1 interactions as a possible strategy for restoration of an anti-HBV immune response.
      • Liu J.
      • Zhang E.
      • Ma Z.
      • et al.
      Enhancing virus-specific immunity in vivo by combining therapeutic vaccination and PD-L1 blockade in chronic hepadnaviral infection.
      Several checkpoint modulators are approved for use in oncology and are thus already available for exploratory studies in CHB patients. One immunologic study concluded that maturation of HBV-specific B cells is adversely affected by the presence of serum HBsAg regardless of disease stage, and that B-cell maturation could be partially restored by addition of anti-PD1 antibodies.
      • Salimzadeh L.
      • Le Bert N.
      • Dutertre C.A.
      • et al.
      PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.
      However, in a small study of patients with HBeAg-negative CHB, the addition of nivolumab (Bristol-Myers Squibb), an anti-PD1 monoclonal antibody, to GS-4774 (Gilead Sciences), an experimental therapeutic T-cell vaccine, did not significantly impact HBsAg levels except in a single patient, although changes in T-cell and natural killer cell composition were reported.

      Verdon D, Brooks AE, Gaggar A, et al. Immunological assessment of HBeAg-negative chronic hepatitis B patient responses following anti-PD-1 treatment. The Liver Meeting. Washington, DC, October 20–24, 2017.

      Gane EJ, Gaggar A, Nguyen AH, et al. A phase 1 study evaluating anti-PD-1 treatment with or without GS-4774 in HBeAg negative chronic hepatitis B patients. The International Liver Congress. Amsterdam, The Netherlands, April 19–21, 2017.

      The combination of an anti-PD-L1 antibody with siRNA in a woodchuck model led to sustained reductions of HBsAg in some animals.
      • Liu J.
      • Zhang E.
      • Ma Z.
      • et al.
      Enhancing virus-specific immunity in vivo by combining therapeutic vaccination and PD-L1 blockade in chronic hepadnaviral infection.
      Whether combinations of checkpoint inhibitors with alternative regimens will significantly improve efficacy in the clinic remains to be seen.

       Therapeutic Vaccines

      There is an extensive and largely unsuccessful history of therapeutic vaccine development for patients with CHB.
      • Kosinska A.D.
      • Bauer T.
      • Protzer U.
      Therapeutic vaccination for chronic hepatitis B.
      Previous failures have prompted more sophisticated approaches designed to broaden HBV-specific immune responses

      Aguilar JC, Lobaina Y, Penton E, et al. Development of a nasal therapeutic vaccine against chronic hepatitis B. 28th Annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

      and explore combinations with both immunomodulators (anti-PD-1/anti-PDL1) and DAAs (such as siRNA).

      Michler T, Kosinska A, Bunse T, et al. Preclinical study of a combinatorial RNAi/vaccination therapy as a potential cure for chronic hepatitis B. The International Liver Congress. Amsterdam, The Netherlands, April 19–23, 2017.

      Although preclinical work has demonstrated exciting results in nonclinical models, these have yet to be validated in human studies.

      Michler T, Kosinska A, Bunse T, et al. Preclinical study of a combinatorial RNAi/vaccination therapy as a potential cure for chronic hepatitis B. The International Liver Congress. Amsterdam, The Netherlands, April 19–23, 2017.

      • Dembek C.
      • Protzer U.
      • Roggendorf M.
      Overcoming immune tolerance in chronic hepatitis B by therapeutic vaccination.

      Other clinical stage therapeutics of interest

       Nucleic Acid Polymers

      In addition to the development programs mentioned above, several other therapeutic classes are currently being evaluated in the clinic. These therapeutic classes include the nucleic acid polymers being developed by Replicor (REP 2139).
      • Vaillant A.
      Nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis B and hepatitis D infection.
      In patients coinfected with HDV and HBV, REP 2139 in combination with peginterferon alfa-2a provided marked suppression of plasma HDV RNA and HBsAg concentrations; HDV RNA remained undetectable at 1 year after treatment in 7 of the 12 patients, and, after 1.5 to 2 years, HBsAg remained undetectable in 4 patients.
      • Bazinet M.
      • Pântea V.
      • Cebotarescu V.
      • et al.
      Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial.

      Bazinet M, Pantea V, Cebotarescu V, et al. Establishment of persistent functional remission of HBV and HDV infection following REP 2139-Ca and pegylated interferon alpha-2a therapy in patients with chronic HBV/HCV co-infection: 1.5-2 year follow-up results from the REP-301 study. The International Liver Congress. Paris, France, April 11–15, 2018.

      In a subsequent study, patients with HBeAg-negative CHB were treated for 48 weeks with combinations of REP 2139 or REP 2165 and both tenofovir and peginterferon alfa-2a.

      Bazinet M, Pantea V, Placinta G, et al. Establishment of high rates of functional control and reversal of fibrosis following treatment of HBeAg negative chronic HBV infection with REP 2139-Mg/REP 2165-Mg, tenofovir disoproxil fumarate and pegylated interferon alpha-2a. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      Bazinet M, Pantea V, Placinta G, et al. Updated follow-up analysis in the REP 401 protocol: treatment of HBeAg negative chronic HBV infection with REP 2139-Mg or REP 2165-Mg, tenofovir disoproxil fumarate and pegylated interferon alfa-2a. The International Liver Congress. Paris, France, April 11–15, 2018.

      At 24 to 48 weeks of posttreatment follow-up, 14 of 34 patients had undetectable HBsAg and HBV DNA; significant alanine aminotransferase flares were common and were correlated with antiviral responses. Positive results have also been reported in an uncontrolled study conducted in Bangladesh.
      • Al-Mahtab M.
      • Bazinet M.
      • Vaillant A.
      Safety and efficacy of nucleic acid polymers in monotherapy and combined with immunotherapy in treatment-naive Bangladeshi patients with HBeAg+ chronic hepatitis B infection.
      A different approach is being taken by Spring Bank, which is developing inarigivir (SB9200), an orally administered linear dinucleotide, as an RIG-I agonist with the objective of activating cellular innate immune responses in HBV-infected cells.
      • Sato S.
      • Li K.
      • Kameyama T.
      • et al.
      The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.
      Inarigivir is also a relatively weak inhibitor of the HBV polymerase.

      College D, Jackson K, Sozzi V, et al. Inarigivir is a novel selective inhibitor of the HBV replicase complex in vitro. The Liver Meeting. San Francisco, CA, November 9–13, 2018.

      After 12 weeks of dosing in patients with CHB, inarigivir elicited modest declines in both HBV DNA and HBV RNA, with somewhat greater efficacy reported in HBeAg-negative versus HBeAg-positive patients at doses up to 100 mg.

      Locarnini S, Wong D, Jackson K, et al. Novel antiviral activity of SB 9200 (inarigivir), a RIG-I agonist: results from cohort 1 of the ACHIEVE trial. The Liver Meeting. Washington, DC, October 20–24, 2017.

      Yuen MF, Elkashab M, Chen CY, et al. Dose response and safety of the daily, oral RIG-I agonist inarigivir (SB 9200) in treatment naïve patients with chronic hepatitis B: results from the 25 mg and 50 mg cohorts in the ACHIEVE trial. The International Liver Congress. Paris, France, April 11–15, 2018.

      Yuen MF, Elkhashab M, Chen CY, et al. Predictors of inarigivir dose response in HBV treatment-naive patients: role of HBeAg status and baseline HBsAg in antiviral response. 28th Annual Conference of the Asian Pacific Association for the Study of the Liver. Manila, Philippines, February 20–24, 2019.

      Data at higher doses are anticipated in 2019. A collaborative study with Gilead Sciences is evaluating inarigivir in combination with tenofovir alafenamide in adults with CHB; other phase 2 and phase 3 combination studies are planned.

       Farnesoid X Receptor Agonists

      The gene encoding the NTCP receptor, which mediates HBV entry into hepatocytes, contains 2 farnesoid X receptor (FXR) α response elements.
      • Radreau P.
      • Porcherot M.
      • Ramiere C.
      • et al.
      Reciprocal regulation of farnesoid X receptor alpha activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.
      FXR agonists reportedly inhibit HBV replication, and at least 1 FXR agonist (EYP001, Enyo Pharma) is entering phase 2 evaluation in patients with CHB, based on preclinical data, suggesting potential to suppress both HBsAg and HBeAg production.

      Joly S, Porcherot M, Radreau P, et al. The selective FXR agonist EYP001 is well tolerated in healthy subjects and has additive anti-HBV effect with nucleoside analogues in HepaRG cells. The International Liver Congress. Amsterdam, The Netherlands, April 19–23, 2017.

      Future therapies (not yet in clinical development)

      In addition to the studies in clinical development, a growing number of targets are being explored preclinically.

       PAPD5/7 Inhibition

      Dihydroquinolizinones have been identified as small molecules that can reduce production of both viral DNA and viral antigens.
      • Mueller H.
      • Lopez A.
      • Tropberger P.
      • et al.
      PAPD5/7 are host factors that are required for hepatitis B virus RNA stabilization.
      Elucidation of the target for these agents determined that they inhibit the catalytic domain of 2 enzymes, PAPD5 and PAPD7, leading to destabilization of HBV mRNA without impacting production of transcripts. Although this target has accrued significant interest, no compounds of this type have yet progressed to studies in patients with CHB.

       RNAse H

      RNAse H activity is required for production of new infectious virus and replenishment of nuclear cccDNA through conversion of rcDNA into cccDNA. Ablating HBV RNase H activity causes accumulation of long RNA:DNA heteroduplexes, truncates minus-polarity DNA strands, and blocks production of the plus-polarity DNA strand.
      • Tavis J.E.
      • Lomonosova E.
      The hepatitis B virus ribonuclease H as a drug target.
      Several chemical leads have been identified, but they also have not yet entered clinical evaluation.
      • Cai C.W.
      • Lomonosova E.
      • Moran E.A.
      • et al.
      Hepatitis B virus replication is blocked by a 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) inhibitor of the viral ribonuclease H activity.
      • Edwards T.C.
      • Mani N.
      • Dorsey B.
      • et al.
      Inhibition of HBV replication by N-hydroxyisoquinolinedione and N-hydroxypyridinedione ribonuclease H inhibitors.
      • Long K.R.
      • Lomonosova E.
      • Li Q.
      • et al.
      Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice.

       Epigenetic Modifiers

      Epigenetic regulation of HBV has recently been extensively reviewed.
      • Hong X.
      • Kim E.S.
      • Guo H.
      Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B.
      “Epigenetics” refers to (heritable) alterations in gene expression that occur to a chromosome without changes in DNA sequence. Because HBV cccDNA exists as a nucleosome-decorated minichromosome, replete with histones and other host proteins, it can presumably be regulated by small-molecule epigenetic-modifying agents, such as those that target histone deacetylases (HDAC), histone acetyltransferases, methyltransferases, and demethylases (KDMs). Indeed, HDAC inhibitors have been shown to suppress cccDNA transcription in tissue culture under noncytotoxic conditions.
      • Yu H.B.
      • Jiang H.
      • Cheng S.T.
      • et al.
      AGK2, a SIRT2 inhibitor, inhibits hepatitis B virus replication in vitro and in vivo.
      Interestingly, transcription from HBV DNA integrated into the host genome was enhanced, suggesting that cccDNA is regulated differently than transcription from an integrated genome.
      • Liu F.
      • Campagna M.
      • Qi Y.
      • et al.
      Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes.
      As HDAC inhibitors have been approved for other indications, it may be anticipated that these could be used in patients with CHB as well, perhaps in combination with an immunotherapeutic, should they be useable at a dose that is free of toxicity. Other epigenetic approaches have also been contemplated, and at least 1 company (Gilead Sciences) has explored targeting KDM5, demonstrating potent reductions of viral antigens as well as RNA associated with histone demethylation (H3K4me3:H3)

      Gilmore SA, Tam D, Dick R, et al. Antiviral activity of GS-5801, a liver-targeted prodrug of a lysine demethylase-5 inhibitor, in a hepatitis B virus primary human hepatocyte infection model. The International Liver Congress. Amsterdam, The Netherlands, April 19–23, 2017.

      ; however, future development for this target indication is uncertain.

       Direct cccDNA Targeting

      The ability to directly eliminate or silence HBV cccDNA has been considered the “holy grail” of HBV therapies. However, until recently, such direct targeting of the viral reservoir has not been feasible. The emergence of CRISPR-Cas9 and other technologies that can directly edit DNA (zinc finger nucleases and such) has the potential to target cccDNA directly. Several companies have emerged around this exciting platform, and in nonclinical studies, several academic groups have shown that this approach can successfully reduce functional cccDNA.
      • Kennedy E.M.
      • Bassit L.C.
      • Mueller H.
      • et al.
      Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease.
      • Kennedy E.M.
      • Kornepati A.V.
      • Cullen B.R.
      Targeting hepatitis B virus cccDNA using CRISPR/Cas9.
      • Lin S.R.
      • Yang H.C.
      • Kuo Y.T.
      • et al.
      The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo.
      • Weber N.D.
      • Stone D.
      • Sedlak R.H.
      • et al.
      AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.
      Although the data are exciting, several important caveats remain: (1) elimination of off-target effects need to be addressed; (2) the vector for delivery would need to access all infected hepatocytes to remove the potential for reactivation; and (3) although studies have shown that integrated genomes may be targeted, cleavage of such integrants runs the theoretical risk of inducing genomic instability, with the concomitant risk of carcinogenesis. It remains to be seen whether these questions can be addressed before entry into the clinic.

      Summary

      The last several years have seen a dramatic resurgence of interest in HBV therapeutic research, with multiple novel targets beyond the HBV pol/RT being explored for the first time in almost 20 years. Although it is early, it is anticipated that the next several years will yield important new insights into the underlying biology of HBV in parallel with new treatments that may have the potential to cure substantially more patients than can be achieved today.

      Acknowledgments

      The author gratefully acknowledges Jason Deer, MS, for his help, and apologizes to numerous colleagues whose inputs and original contributions could not be included or cited due to space and topical constraints. The author also gratefully acknowledges the editorial assistance provided by Richard Boehme, PhD, of MediTech Media, and funding by Assembly Biosciences .

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